CN105969764A - Method for extracting DNA from animal tissues - Google Patents
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- CN105969764A CN105969764A CN201610556689.5A CN201610556689A CN105969764A CN 105969764 A CN105969764 A CN 105969764A CN 201610556689 A CN201610556689 A CN 201610556689A CN 105969764 A CN105969764 A CN 105969764A
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Abstract
The invention relates to the technical field of biology, particularly a method for extracting DNA from animal tissues. The method comprises the following steps: carrying out lysis on animal tissues to obtain a lysis mixed solution; adding a salt-carbon mixed solution into the lysis mixed solution, uniformly mixing, and carrying out ice bath treatment and centrifugation to obtain a supernate; adding isopropanol into the supernate, and uniformly mixing to obtain a flocculent precipitate mixed solution; and carrying out centrifugation on the flocculent precipitate mixed solution to obtain the DNA precipitate. By using nontoxic NaCl and activated carbon instead of phenol, chloroform and other toxic reagents in the existing process, the method provided by the invention lowers the safety risk in the animal tissue DNA extraction process. The fundamental principle for extraction is to implement the DNA-impure substance separation by utilizing the solubility difference between DNA and other substances under high-salinity conditions and the adsorptivity of the activated carbon.
Description
Technical field
The present invention relates to biological technical field, carry in particular to a kind of animal tissue
Take DNA method.
Background technology
DNA (being the abbreviation of English Deoxyribonucleic acid), also known as deoxyribose
Nucleic acid, is the main chemical compositions of chromosome, is also simultaneously genomic constitution, sometimes by
It is referred to as " hereditary particle ".DNA is a kind of molecule, can form genetic command, raw to guide
Thing is grown and is operated with vital functions.Major function is that the information of chronicity stores, and can liken
For " blueprint " or " recipe ".The instruction wherein comprised, is construction other change intracellular
Compound, needed for protein and RNA.DNA fragmentation with hereditary information is referred to as base
Cause, other DNA sequence, some directly plays a role with construction itself, and some is then joined
Performance with regulation and control hereditary information.
DNA as one of hereditary material in organism, be widely present in animal, plant,
In microorganism, it is the important experiment material of molecular biology research, raw at the molecule of animal
Thing research has, generally requires the DNA extracting animal tissue.
Although the method for animal tissue's DNA extraction has a lot now, such as phenol chloroform
Method, complex operation, generally require and repeatedly extract, and operating process is repeatedly used chlorine
The multiple poisonous organic reagents such as imitative, phenol；High salt CTAB method, although this method behaviour
Make simple, but be used for the extraction of DNA in plant tissue, for pluck tissue
The success rate extracted is relatively low, and the method impurity in products is more.
In view of this, this invention of special proposition.
Summary of the invention
It is an object of the invention to provide a kind of method for extracting DNA from animal tissue, this method
Mainly utilize dissolubility difference and the activated carbon of DNA and other materials under the conditions of high salinity
Adsorption, it is achieved DNA separates with impurity substances, and this method not only operates
Simply, success rate is high, product quality is good.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
A kind of animal tissue extracts DNA method, comprises the steps:
After animal tissue being cracked, obtain cracking mixed liquor；
In described cracking mixed liquor add salt-charcoal mixed liquor, after mixing successively ice bath, from
The heart, obtains supernatant；
In described supernatant, add isopropanol, after mixing, obtain flocculent deposit mixed liquor；
DNA precipitation is obtained after centrifugal for described flocculent deposit mixed liquor.
The present invention use method for extracting DNA from animal tissue, utilize nontoxic NaCl and
Activated carbon replaces the toxic reagents such as the phenol in existing method, chloroform, reduces extraction dynamic
Security risk during fabric texture DNA extraction；The ultimate principle that the present invention extracts is profit
With DNA under the conditions of high salinity with dissolubility difference and the suction of activated carbon of other materials
Attached effect, it is achieved DNA separates with impurity substances.
Preferably, cleavage method is: add lysate and protease in animal tissue,
Constant temperature oscillation 2～3Hr under the conditions of 50 DEG C～60 DEG C, obtains cracking mixed liquor.
In method for extracting DNA from animal tissue, huge genomic DNA is to be easy to
Snarl the protein of macromole, the macro-molecular protein right and wrong of DNA are enwinded for these
Often it is difficult to remove, the DNA purity that impact is extracted；Therefore, the DNA that this patent provides
In extracting method, the effect of lysate is to destroy cell membrane, nuclear membrane, and makes histone
Separate with DNA, the activity of deoxyribonuclease in suppression cell；The effect of protease is
The protein making macromole diminishes, and free to protein has huge facilitation.When
After protein is diminished by protease digestion, then it is not easy to be snarled, favorably by genomic DNA
In protein removal in purge process, make the purity of DNA finally obtained higher.
The method is the most simple to operate, success rate is high, product quality is good, and solves existing
Method for extracting DNA from animal tissue in the halfway problem of Tissue Lysis, improve extraction
Efficiency.
In the method for extracting DNA from animal tissue that the present invention uses, lysate and protease
Select all to affect the success rate of extraction and final product purity.
Preferably, in every 1 liter of described lysate, containing 20ml 1M Tris-HCl
(pH8.0), 10ml 0.5M EDTA, 23.376g NaCl, 100ml 10%SDS, remaining
Amount is distilled water.
It is highly preferred that described protease is E.C. 3.4.21.64.
Through multiple authentication, use above-mentioned lysate and E.C. 3.4.21.64, make animal tissue split
Solution is very thorough, adds extracting method success rate, product quality good, and improves
The efficiency extracted.
Preferably, in described cracking mixed liquor, salt-charcoal mixed liquor, mixing are added described
After ice bath, centrifugal successively, obtain in the step of supernatant:
Described centrifugal process is to be centrifuged 10～20min under the conditions of 12000rpm；
Described ice bath process is 10～20min.
In the method for extracting DNA from animal tissue that this patent uses, ice bath process is to make
Insoluble protein precipitates, and keeps the ice bath time of 10～20min to substantially carry out
The precipitation of major part insoluble protein, by centrifugal the 10～20min of 12000rpm,
Achieve precipitation and the separation of supernatant, improve the product quality of final DNA.
It is highly preferred that for ensureing to extract the DNA of better quality, centrifugal process be
Centrifugal 15min under the conditions of 12000rpm；
Ice bath process is 15min.
Preferably, also include after obtaining described flocculent deposit mixed liquor: by described wadding
Shape precipitation mixed liquor places 1～3Hr under the conditions of-15 DEG C～-25 DEG C；
Or described flocculent deposit mixed liquor is placed under the conditions of 0 DEG C～6 DEG C 8～16Hr.
The present invention use method for extracting DNA from animal tissue in, add isopropanol it
Rear it is possible that flocculent deposit is less or occurs without the phenomenon of flocculent deposit, therefore will wadding
Shape precipitation mixed liquor places 1～3Hr or by described wadding under the conditions of being positioned over-15 DEG C～-25 DEG C
Shape precipitation mixed liquor places 8～16Hr under the conditions of 0 DEG C～6 DEG C, under these conditions can
Enough make the precipitation completely of flocky precipitate.
More preferably, for increasing the precipitation of flocky precipitate, by flocculent deposit mixed liquor
2Hr is placed under the conditions of being positioned over-20 DEG C；
Or described flocculent deposit mixed liquor is placed under the conditions of 4 DEG C 12Hr.
Preferably, also include after obtaining described DNA precipitation:
Described DNA precipitation is dissolved completely in TE buffer, obtains DNA and store liquid.
TE buffer is a kind of solution that can resist pH change when adding a small amount of acid or alkali.
The PH buffer system normal ph to maintenance biology, normal physiological context plays an important role.
Most cells are only capable of in the range of the narrowest pH carrying out activity, and need buffer system
Resist the pH change occurred in metabolic process.Therefore, the animal used at this patent
In tissue DNA extracting method, use TE buffer solution DNA, play storage-stable
The purpose of DNA.
Having three kinds of main pH buffer systems in organism, they are protein, weight carbon
Hydrochlorate buffer system and phosphate buffer.Component shared by every kind of buffer system is respectively
Class cell and organ are different；Therefore, those skilled in the art can be according to difference
Animal tissue select different TE buffer.
Preferably, also include after obtaining described DNA precipitation:
It is centrifuged after adding alcohol flushing in described DNA precipitates, air-dries after absorbing ethanol,
Obtain refined DNA precipitation.
In the method for extracting DNA from animal tissue that this patent uses, described flocculent deposit is mixed
Close the DNA precipitation obtained after liquid is centrifuged and also may can comprise a part of impurity, such as isopropyl
Alcohol.Therefore in described DNA precipitates, add ethanol be rinsed, and use rifle head by second
Alcohol air-dries after absorbing, and i.e. can obtain refined DNA.Therefore, DNA precipitation is clear through ethanol
Absorb after washing and air-dry, substantially can occasionally remove the isopropanol of residual in DNA precipitation, make
The refined DNA precipitated product quality finally given is high.
Preferably, it is centrifuged after addition alcohol flushing in DNA precipitates described, absorbs second
Air-dry after alcohol, obtain in the step of refined DNA precipitation:
Described centrifugal process is to be centrifuged 0.5～2min under the conditions of 12000rpm.
Preferably, add during described salt-charcoal mixed liquor is the saturated NaCl solution of every 30ml
1g activated carbon is mixed to get.
Compared by prior art, the DNA extraction side of a kind of animal tissue that the present invention provides
Method, utilizes dissolubility difference and the activated carbon of DNA and other materials under the conditions of high salinity
Adsorption, it is achieved DNA separates with impurity substances, this method is the most simple to operate,
Success rate is high, and the product quality obtained is good.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and not
Should be regarded as limiting the scope of the present invention.Unreceipted actual conditions person in embodiment, according to often
The condition of rule condition or manufacturer's suggestion is carried out.Agents useful for same or the unreceipted factory of instrument
Shang Zhe, being can be by the conventional products of commercially available acquisition.
Embodiment 1
Technical scheme effect, the present embodiment 1 is used to provide existing skill by explanation this patent
Method for extracting DNA from animal tissue phenol chloroform extraction method in art, its specific experiment mistake
Journey is as follows:
1), take tissue sample shredding to be placed in 1.5mL centrifuge tube；
2), in centrifuge tube, Tissue lysates and E.C. 3.4.21.64 mixed liquor are added, at 37 DEG C
12Hr is placed under water bath condition；
3), in centrifuge tube, add balance phenol 500uL, fully mix, 4 DEG C, 12000rpm
Under the conditions of centrifugal 15min；
4), careful upper strata aqueous phase of drawing is in another centrifuge tube；
5), step 3 and 4 is repeated；
6), in centrifuge tube, add chloroform-isoamyl alcohol (ratio 24:1) 500uL, fully mix,
4 DEG C, centrifugal 15min under the conditions of 12000rpm, careful draw upper strata aqueous phase to another from
In heart pipe
7), step 6 is repeated until can't see protein gel；
8), accurately measure supernatant, and transfer to, in new centrifuge tube, add in centrifuge tube
Enter the cold ethanol of 2 times of volumes, place 1Hr for 4 DEG C, it is seen that cotton-shaped DNA separates out；
9), at 4 DEG C, under the conditions of 12000rpm centrifugal 15 minutes, DNA precipitation is obtained,
Discard supernatant；
10), to above-mentioned DNA precipitate in add 500uL70% ethanol, 12000rpm from
The heart 10 minutes, abandons supernatant, is repeated twice；
11), natural air drying DNA precipitates 10 minutes；
12), in DNA precipitates, 100uL TE buffer or distilled water are added, at 4 DEG C
Under the conditions of 12Hr make DNA resolution of precipitate；Properly preserve this DNA and store liquid.
Phenol chloroform extraction method of the prior art, uses balance phenol and chloroform to animal tissue
Process, and need to carry out operation is repeated several times；Balance phenol and chloroform have bigger
Toxicity, therefore using during phenol chloroform extraction method carries out animal tissue's DNA extraction,
Pay particular attention to experiment safety problem.
Embodiment 2
1), clip animal tissue shredding, put it in the centrifuge tube of 1.5mL；
2), in centrifuge tube, 500uL lysate and 20uL E.C. 3.4.21.64 (both are added
Can be the most mixed), centrifuge tube is positioned in constant temperature oscillator 55 DEG C and vibrates 2 hours；
3), take out the sufficient centrifuge tube of cracking, in often pipe, add 300uL salt-charcoal
Mixed liquor (with front concussion mixing), 6-8 rear ice bath of reverse mixing 15 minutes；
4), it is centrifuged 15 minutes under 12000rpm, room temperature condition, takes carefully
In the Axgen centrifuge tube of clear 500uL to new 1.5mL (during drawing carefully
Avoid being drawn onto precipitation)；
5), isopyknic 500uL isopropanol is added in Axgen centrifuge tube, reverse
Until formation flocculent deposit；
6), after 12000rpm, room temperature condition are centrifuged 15 minutes, abandon supernatant, obtain
DNA precipitates.
In a kind of method for extracting DNA from animal tissue that the present embodiment provides, utilize nontoxic
NaCl and activated carbon replace the toxic reagents such as the phenol in existing method, chloroform, reduce
Extract the security risk during animal tissue's DNA extraction；It is the most former that the present invention extracts
Reason be utilize DNA under the conditions of high salinity with dissolubility difference and the activity of other materials
The adsorption of charcoal, it is achieved DNA separates with impurity substances.
In the present embodiment, the preparation method of lysate is: by 20ml 1M Tris-HCl
(pH8.0), 10ml 0.5M EDTA, 23.376g NaCl, 100ml 10%SDS mix
After conjunction, it is settled to 1L with distilled water.For ensureing the quality of final extract in this patent,
This lysate needs to carry out autoclave sterilization process before use；
In the present embodiment, the final utilization concentration of E.C. 3.4.21.64 is 0.4mg/ml；This egg
White enzyme K is it is generally required to preserve under the conditions of-20 DEG C, and is preparing the mistake of this E.C. 3.4.21.64
The solvent used in journey is sterilized water.
In the present embodiment, the preparation method of salt-charcoal mixed liquor is: every saturated NaCl of 30ml
Solution adds 1g activated carbon and mixes, and wherein activated carbon is preferably powdered active carbon；
On the one hand powdered active carbon can increase adsorption area, improves adsorption efficiency so that miscellaneous
The absorption of matter is the most complete.
In method for extracting DNA from animal tissue, huge genomic DNA is to be easy to
Snarl the protein of macromole, the macro-molecular protein right and wrong of DNA are enwinded for these
Often it is difficult to remove, the DNA purity that impact is extracted；Therefore, the present embodiment provides
In DNA extraction method, the effect of lysate is to destroy cell membrane, nuclear membrane, and makes tissue
Albumen separates with DNA, the activity of deoxyribonuclease in suppression cell；E.C. 3.4.21.64
Effect is that the protein making macromole diminishes, and free to protein has huge promotion to make
With.After protein is diminished by protease digestion, then it is not easy to be snarled by genomic DNA,
Be conducive to protein removal in purge process, make the purity of DNA that finally obtains more
High.The DNA extraction method that the present embodiment provides is the most simple to operate, success rate is high, product
Material amount is good, and solves Tissue Lysis in existing method for extracting DNA from animal tissue
Halfway problem, improves extraction efficiency.
Embodiment 3
1), clip animal tissue shredding, put it in the centrifuge tube of 1.5mL；
2), in centrifuge tube, 500uL lysate and 20uL E.C. 3.4.21.64 (both are added
Can be the most mixed), centrifuge tube is positioned in constant temperature oscillator 60 DEG C and vibrates 2.5 hours；
3), take out the sufficient centrifuge tube of cracking, in often pipe, add 300uL salt-charcoal
Mixed liquor (with front concussion mixing), 6-8 rear ice bath of reverse mixing 20 minutes；
4), it is centrifuged 20 minutes under 12000rpm, room temperature condition, takes carefully
In the Axgen centrifuge tube of clear 500uL to new 1.5mL (during drawing carefully
Avoid being drawn onto precipitation)；
5), isopyknic 500uL isopropanol is added in Axgen centrifuge tube, reverse
Until formation flocculent deposit；
6), after 12000rpm, room temperature condition are centrifuged 20 minutes, abandon supernatant, obtain
DNA precipitates.
7), in DNA precipitates, 100uL TE buffer or distilled water are added, at 4 DEG C
Under the conditions of 12Hr make DNA resolution of precipitate；Properly preserve this DNA and store liquid.
In the present embodiment, use TE buffer solution DNA, play storage-stable DNA
Purpose；TE buffer is a kind of to change by opposing pH when adding a small amount of acid or alkali
Solution.The PH buffer system normal ph to maintenance biology, normal physiological context lifting
Act on.Most cells are only capable of in the range of the narrowest pH carrying out activity, and need
Buffer system resists the pH change occurred in metabolic process.
In addition, it is necessary to explanation, organism has three kinds of main pH buffer systems,
They are protein, heavy carbonate buffer system and phosphate buffer.Every kind of buffering
Component shared by system is different in various types of cells and organ；Therefore, this area skill
Art personnel can select different TE buffer according to different animal tissues.
Embodiment 4
1), clip animal tissue shredding, put it in the centrifuge tube of 1.5mL；
2), in centrifuge tube, 500uL lysate and 20uL E.C. 3.4.21.64 (both are added
Can be the most mixed), centrifuge tube is positioned in constant temperature oscillator 50 DEG C and vibrates 2 hours；
3), take out the sufficient centrifuge tube of cracking, in often pipe, add 300uL salt-charcoal
Mixed liquor (with front concussion mixing), 6-8 rear ice bath of reverse mixing 10 minutes；
4), it is centrifuged 10 minutes under 12000rpm, room temperature condition, takes carefully
In the Axgen centrifuge tube of clear 500uL to new 1.5mL (during drawing carefully
Avoid being drawn onto precipitation)；
5), isopyknic 500uL isopropanol is added in Axgen centrifuge tube, reverse
Until formation flocculent deposit；
6), by step 5) under the conditions of the flocculent deposit mixed liquor that obtains is positioned over-20 DEG C
Place 2Hr.
7), after 12000rpm, room temperature condition are centrifuged 10 minutes, abandon supernatant, obtain
DNA precipitates.
Embodiment 5
In the present embodiment, except step 6) in addition to other step the most the same as in Example 4,
Wherein step 6) implementation process be:
6), by step 5) in the flocculent deposit mixed liquor that obtains place under the conditions of 4 DEG C
12Hr。
In embodiment 4 with embodiment 5, it is possible that wad a quilt with cotton after adding isopropanol
Shape precipitates phenomenon that is less or that occur without flocculent deposit, therefore uses and puts under cryogenic
Put certain time, to reach the precipitation completely of flocky precipitate.
Embodiment 6
On the basis of embodiment 2, also comprise the steps:
7), add 70% ethanol of 500uL in each pipe, reverse rinse precipitation；
8), 12000rpm be centrifuged 1 minute, with the rifle head of 200uL, ethanol is sopped up,
Refined DNA is stayed to be deposited in pipe (the most not siphoning away refined DNA precipitation)；
9), natural air drying refines DNA precipitation.
In the method for extracting DNA from animal tissue provided in the present embodiment, by described cotton-shaped heavy
The DNA precipitation obtained after shallow lake mixed liquor is centrifugal also may can comprise a part of impurity, such as
Isopropanol.Therefore in described DNA precipitates, add ethanol be rinsed, and use rifle head
Air-dry after ethanol is absorbed, i.e. can obtain refined DNA.Therefore, DNA precipitates through second
Alcohol is absorbed and is air-dried after cleaning, and substantially can remove except the isopropanol of residual in DNA precipitation,
Make that the refined DNA precipitated impurities finally given is few, quality is high.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that
May be made that other changes many without departing from the spirit and scope of the present invention
And amendment.It is, therefore, intended that include in the following claims belonging to the scope of the invention
Interior all such changes and modifications.
Claims (10)
1. an animal tissue extracts DNA method, it is characterised in that comprise the steps:
After animal tissue being cracked, obtain cracking mixed liquor；
Salt-charcoal mixed liquor is added in described cracking mixed liquor, ice bath, centrifugal successively after mixing, obtain supernatant
Liquid；
In described supernatant, add isopropanol, after mixing, obtain flocculent deposit mixed liquor；
DNA precipitation is obtained after centrifugal for described flocculent deposit mixed liquor.
Animal tissue the most according to claim 1 extracts DNA method, it is characterised in that described cracking
Method is:
Addition lysate and protease in animal tissue, constant temperature oscillation 2～3Hr under the conditions of 50 DEG C～60 DEG C,
Obtain cracking mixed liquor.
Animal tissue the most according to claim 2 extracts DNA method, it is characterised in that every 1 liter of institute
State in lysate, containing 20ml 1M Tris-HCl (pH8.0), 10ml 0.5M EDTA, 23.376g
NaCl, 100ml 10%SDS, surplus is distilled water.
Animal tissue the most according to claim 2 extracts DNA method, it is characterised in that described albumen
Enzyme is E.C. 3.4.21.64.
Animal tissue the most according to claim 1 extract DNA method, it is characterised in that described to
Described cracking mixed liquor adds salt-charcoal mixed liquor, ice bath, centrifugal successively after mixing, obtain supernatant
Step in:
Described centrifugal process is to be centrifuged 10～20min under the conditions of 12000rpm；
Described ice bath process is 10～20min.
Animal tissue the most according to claim 1 extracts DNA method, it is characterised in that obtaining
Also include after stating flocculent deposit mixed liquor: by described flocculent deposit mixed liquor-15 DEG C～-25 DEG C of conditions
Lower placement 1～3Hr；
Or described flocculent deposit mixed liquor is placed under the conditions of 0 DEG C～6 DEG C 8～16Hr.
7. extract DNA method according to the animal tissue described in claim 1-6, it is characterised in that obtaining
Also include after described DNA precipitation:
Described DNA precipitation is dissolved completely in TE buffer, obtains DNA and store liquid.
8. extract DNA method according to the animal tissue described in claim 1-6, it is characterised in that obtaining
Also include after described DNA precipitation:
It is centrifuged after adding alcohol flushing in described DNA precipitates, air-dries after absorbing ethanol, obtain refined DNA
Precipitation.
Animal tissue the most according to claim 8 extract DNA method, it is characterised in that described to
It is centrifuged after DNA precipitation adds alcohol flushing, air-dries after absorbing ethanol, obtain the step of refined DNA precipitation
In Zhou:
Described centrifugal process is to be centrifuged 0.5～2min under the conditions of 12000rpm.
Animal tissue the most according to claim 1 extracts DNA method, it is characterised in that and described salt-
Charcoal mixed liquor is to add 1g activated carbon in the saturated NaCl solution of every 30ml to be mixed to get.
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